The long-term objective of these experiments is to determine if a particular glycosyltransferase, known as GlcNAc-T V, regulates surface N-linked oligosaccharide expression which can cause a change in the adhesive properties of a cell, and in the case of an oncogenically transformed cell, regulate its metastatic potential. Several lines of evidence suggest that regulation of the oligosaccharides whose expression can be regulated by GlcNAc-T V activity can influence the metastatic potential of cells. Moreover, studies on human breast and colon tissue biopsies have documented a consistent increase in the cell surface product of GlcNAc-T V, the so-called Beta(1,6) branch, in neoplastic disease and a correlation with the pathological staging of the disease. In order to address the hypothesis that GlcNAc-T V activity regulates N- linked oligosaccharide expression on the cell surface which causes alterations in cell adhesion, and ultimately regulation of metastatic potential, we have, in collaboration with Dr. Nevis Fregien, with whom we are submitting this IRPG, obtained a cDNA which encodes the full- length rat GlcNAc-T V. Using this cDNA as a probe, we have now established that transformation of cells with the oncogenes, v-src and neu-her-2 causes an up-regulation of GlcNAc-T V mRNA levels and activity of over 5-fold. Neu-her-2 has been shown to be over-expressed in 30% of human breast carcinomas, and its expression is highly correlated with the number of lymph node metastases, increased probability of tumor recurrence, and reduced patient survival. Therefore, neu-her-2 expression in human breast carcinomas may cause changes in GlcNAc-T V, N-linked oligosaccharide expression, and alteration of cell adhesion. To test this hypothesis, experiments in this proposal are designed to first establish the relationship between GlcNAc-T V activity, mRNA and protein levels. To study the expression of the enzyme protein, we will prepare a polyclonal antibody specific for GlcNAc-T V. We will then generated cell lines whose GlcNAc-T V levels have been modulated by transfection with sense or antisense GlcNAc-T V cDNA constructs. We will use FACS and fluorescent L-PHA to isolate clones with altered cell surface Beta(1,6) branched oligosaccharides. We will then test for the function of these oligosaccharides on various cell adhesion properties by analyzing several adhesive properties of these clones, comparing them with their respective parental cells. We will test the adhesion of cells whose GlcNAc-T V activity has been altered to extracellular matrix proteins and to an endothelial cell line to test if GlcNAc-T V caused cell surface oligosaccharide changes can alter cell adhesive properties. Those cells with altered adhesive properties will then be analyzed in terms of their cell surface N-linked oligosaccharides, as well as their expression of glycoproteins with Beta(1,6) branches and polylactosamines. Finally, in order to test further the function of Beta(1,6) N-linked structures in the adhesion of metastatic cells, we will analyze the adhesive properties of several human metastatic breast cell lines which have been transfected with GlcNAc-T V antisense cDNA to lower cell surface Beta(1 ,6) structures.